A Pilot Study Evaluating Subgenomic RNAs for Detection of Infectious SARS-CoV-2 in Nasopharyngeal Swabs

Background: The prolonged persistence of viral ribonucleic acid (RNA) in coronavirus disease 2019 (COVID-19) patients and the difficulty in differentiating between infectious virus and noninfectious viral RNA have impeded the use of current molecular diagnostic tests as a decision tool in quarantine termination. The performance of new methods to detect surrogate viability markers, such as subgenomic RNAs (sgRNAs), has been discussed, and numerous important questions are still needed to be addressed before broad implementation. Objectives: This study aimed to primarily evaluate the performance of SYBR green quantitative reverse transcription-polymerase chain reaction (RT-qPCR) targeting N and E sgRNAs as a surrogate of viability markers. Methods: This pilot study was carried out to detect genomic RNAs (gRNAs) and sgRNAs using RT-qPCR in cell culture infected with severe acute respiratory syndrome coronavirus 2 and nasopharyngeal swabbing samples from COVID-19 patients, and the results were compared to viral culture as a gold standard method for infectious virus detection. The diagnostic parameters and Cohen’s Kappa correlation index were then analyzed. Results: E subgenomic RNA detection was the most reliable predictor for actively replicating the virus as it showed the highest value of all diagnostic parameters with a good correlation with viral cultivation. The lowest cycle threshold value of gRNAs and sgN detection become undetectable by sgE within the range of 23 - 26. Conclusion: Using a suitable sgRNA type was important for test accuracy. The findings suggested E sgRNA detection as a promising surrogate approach to indicate a truly active viral infection, and when performed with a low-cost molecular test of SYBR green-based assay, it could support huge demands for routine analysis.